The long term objective of this project is to determine the specific roles of the synthetic and hydrolytic enzymes involved in formation of the murein sacculus and the polar caps of gram negative bacteria; a second broad aim is to understand the regulation of these enzymes during the cell division cycle. Currently, 9 of these enzymes are recognized as being sensitive to penicillin and the health relatedness of a study of the metabolism of cell wall murein and the associated enzymes derives from the expectation that increased knowledge of this area of metabolism will aid in development and proper use of penicillin and other beta-lactam antibiotics. One specific aim of this proposal is to complete testing of an hypothesis which may explain how elongation of the sacculus occurs and to identify the penicillin-sensitive enzymes (penicillin-binding proteins) normally involved in the process. The experimental design involves pulse or pulse-chase labeling of cells with diamionpimelic acid and following the formation and fate of the acceptor and donor halves of the cross-linked peptide dimers in the sacculus under various conditions and in appropriate mutants such as, for example, multiple mutant strains to be constructed that contain A-, opp-, one or more penicillin- binding protein mutations, and fts Z. The main thrust of the current proposal will be directed towards the long term objective of determining the specific roles of the penicillin-binding proteins in construction of the murien sacculus. This involves careful study of strains either mutant or selectively inhibited in one or more of the penicillin-binding proteins.